Transgenic Mice Overexpressing Renin Exhibit Glucose Intolerance and Diet-Genotype Interactions.

ENDOCRINOLOGY

ORIGINAL RESEARCH ARTICLE

published: 07 January 2013
doi: 10. 3389/fendo. 2012. 00166

Transgenic mice overexpressing renin exhibit glucose
intolerance and diet-genotype interactions

Sarah J. Fletcher \ Nishan S. Kalupahana 2 , Morvarid Soltani-Bejnood 3 , Jung Han Kim 4 , Arnold M. Saxton 5 ,
David H. Wasserman 6 , Bart DeTaeye 6 , Brynn H. Voy 5 , Annie Quignard-Boulange 7 and
Naima Moustaid-Moussa 8 *

' Genome Science and Technology Program, University of Tennessee, Knoxville, TN, USA

2 Department of Physiology, Faculty of Medicine, University of Peradeniya, Peradeniya, Sri Lanka

3 Pellissippi State, Knoxville, TN, USA

4 Department of Pharmacology, Physiology and Toxicology, School of Medicine, Marshall University, Huntington, WV, USA

5 Department of Animal Science, University of Tennessee, Knoxville, TN, USA

6 Department of Molecular Physiology and Biophysics, School of Medicine and Mouse Metabolic Phenotyping Center, Vanderbilt University, Nashville, TN, USA

7 INRA-AgroParisTech UMH 914, Paris, France

" Nutritional Sciences, Texas Tech University, Lubbock, TX, USA

Edited by:

Tsuguhito Ota, Kanazawa University,
Japan

Reviewed by:

Undurti Narasimha Das, UND Life
Sciences, USA

Torn Aizawa, Aizawa Hospital, Japan

'Correspondence:

Naima Moustaid-Moussa, Nutritional
Sciences, Texas Tech University, 1301,
Akron Street, Lubbock, TX 79423,
USA.

e-mail: naima. moustaid-moussa@
ttu.edu

Numerous animal and clinical investigations have pointed to a potential role of the renin-
angiotensin system (RAS) in the development of insulin resistance and diabetes in condi-
tions of expanded fat mass. However, the mechanisms underlying this association remain
unclear. We used a transgenic mouse model overexpressing renin in the liver (RenTgMK)
to examine the effects of chronic activation of RAS on adiposity and insulin sensitivity.
Hepatic overexpression of renin resulted in constitutively elevated plasma angiotensin II
(four- to six-fold increase vs. wild-type, WT). Surprisingly, RenTgMK mice developed glu-
cose intolerance despite low levels of adiposity and insulinemia. The transgenics also had
lower plasma triglyceride levels. Glucose intolerance in transgenic mice fed a low-fat diet
was comparable to that observed in high-fat fed WT mice. These studies demonstrate that
overexpression of renin and associated hyperangiotensinemia impair glucose tolerance in
a diet-dependent manner and further support a consistent role of RAS in the pathogenesis
of diabetes and insulin resistance, independent of changes in fat mass.

Keywords: adipose tissue, renin-angiotensin system, insulin resistance, angiotensin II

INTRODUCTION

The renin-angiotensin system (RAS) plays an important role in
the regulation of blood pressure, fluid, and electrolyte balance
(Schmieder et al, 2007). Angiotensinogen (AGT), the precursor
peptide of this system, undergoes successive enzymatic cleav-
ages by renin and angiotensin converting enzyme (ACE) to yield
angiotensin I (Ang I) and angiotensin II (Ang II) respectively.
The latter is the main bioactive peptide of this system, which acts
via two G-protein coupled receptors, namely angiotensin Type-
1 (ATI) and Type-2 (AT2) receptors, to exert its physiological
effects. Because ATI activation by Ang II leads to elevation of blood
pressure, ACE inhibitors (ACEI) and ATI blockers (ARB) are phar-
macologically used as anti-hypertensive agents (Schmieder et al.,
2007).

Interestingly, several clinical studies have shown that patients
on RAS blockers have a lower risk of developing Type-2 diabetes
when compared to patients on other anti-hypertensive medica-
tions (Vermes et al, 2003; Bosch et al, 2006). Moreover, RAS
blockade prevents and reverses insulin resistance induced by high-
fat feeding in rodents (Lee et al, 2008). Given that plasma and
tissue levels of several RAS components positively correlate with
body mass index (Schorr et al., 1998; Van Harmelen et al., 2000),
it is possible that overactivation of the RAS is linked to the patho-
genesis of insulin resistance in obesity. Indeed, genetic deletion of

AGT, ACE, renin, ATI, or AT2 protects rodents from diet-induced
obesity and insulin resistance (Massiera et al., 200 lb; Yvan-Charvet
et al, 2005; Takahashi et al, 2007; Jayasooriya et al, 2008). Con-
versely, chronic RAS overactivation via Ang II infusion (Ogihara
et al., 2002) leads to glucose intolerance and insulin resistance in
rodents, further supporting a role of RAS overactivation in the
pathogenesis of insulin resistance.

Although obesity and increased adiposity are associated with
RAS overactivation, it is not clear whether systemic RAS over-
activation can lead to both obesity and insulin resistance. It is
important to test this because studies have documented differ-
ences in RAS activity in humans, which have been attributed to
polymorphisms in RAS coding (Jeunemaitre et al., 1999; Jeune-
maitre, 2008) or promoter regions (Xiao et al., 2006). Therefore,
understanding the implications of chronic elevation of RAS may
help provide insight into metabolic consequences of chronically
elevated RAS in humans.

While overexpression of RAS is consistently associated with
insulin resistance and glucose intolerance, the effect of chronic
RAS overactivation on adiposity is not clear. This is further com-
plicated by existence of local RAS in several tissues with the local
effects complicating the understanding of systemic effects of RAS
(Kalupahana and Moustaid-Moussa, 2012b). For example, over-
expression of AGT in adipose tissue increases adiposity and blood

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Metabolic phenotyping of renin transgenic mice

pressure and leads to insulin resistance (Massiera et al., 2001a;
Kalupahana et al., 2012). However, acute or chronic systemic RAS
overactivation leads to decreased fat mass despite the develop-
ment of insulin resistance (Brink et al., 1996). This suggests that
increased fat mass in the case of adipose RAS overexpression
may be due to local effects of Ang II production within adipose
tissue.

To further dissect effects of elevated systemic Ang II on insulin
sensitivity and adiposity, we used a unique mouse model in
which Ang II is chronically elevated throughout life time through
genetic manipulation. This mouse model is a unique genetic
minipump model in which renin is overexpressed in the liver.
Given that renin release is the rate-limiting step in the sys-
temic RAS, this model offers the advantage of constant renin
overexpression independent of homeostatic control and a life-
long elevated level of Ang II. As expected, these transgenic mice
(RenTgMK; Caron et al., 2002) exhibit elevated levels of circu-
lating renin and Ang I and develop chronic hypertension along
with other pathological manifestations (Caron et al., 2002, 2004).
The RenTgMK mice thus allow us also to study the effects of
systemic chronic elevations of Ang II on adiposity and glucose
homeostasis, so that we can dissect the effects of systemic vs. adi-
pose RAS by comparing these results with the ones previously
reported for local adipose overexpression of RAS (Massiera et al.,
2001a).

We report here that elevated circulating Ang II due to renin
overexpression leads to glucose intolerance, which is further exac-
erbated by high-fat feeding. Unexpectedly, these mice exhibit
otherwise normal glucose metabolism and a transgene dose-
dependent decrease in fat mass and insulinemia.

MATERIALS AND METHODS
ANIMALS

RenTgMK transgenic mice were kindly provided by Dr. Oliver
Smithies, University of North Carolina, Chapel Hill, NC, USA
(Hatada et al., 1999). Briefly, a renin transgene consisting of por-
tions of the Ren2 and Ren- 1 d genes (Ren2/ 1 d) was inserted into the
genome at the ApoAl/ApoC3 locus via homologous recombina-
tion and placed under control of an albumin promoter/enhancer
(AlbP/E) to achieve liver-specific expression.

Male heterozygous RenTgMK (RenTgMK~ /+ ) mice on an iso-
genic SvEv 129/6 background were crossed with wild-type (WT)
SvEv females. Subsequent heterozygous Fl progeny were mated to
generate the F2 offspring that were used in this study. Mice used
in this study were bred and maintained at the University of Ten-
nessee accredited animal facility, on a 12h:12h light-dark cycle at
22°C and fed a standard rodent chow and water ad libitum. All
experiments were approved by the Institutional Animal Care and
Use Committee at the University of Tennessee.

GENOTYPING

DNA was extracted from tail tips as previously described
(Truett et al, 2000). PCR-based genotyping was performed
using three primers: pi, 5'-TGGGATTCTAACCCTGAGGACC-
3'; p2, 5'-CACAGATTGTAACTGCAAATCTGTCG-3'; p3, 5'-
GTTCTTCTGAGGGGATC-GGC-3' (Sigma Genosys, The Wood-
lands, TX, USA) as previously described (Caron et al., 2002).

GLUCOSE TOLERANCE TEST

Mice were fasted overnight prior to the glucose tolerance test
(GTT). Blood was collected in heparinized capillary tubes from
the orbital sinus prior to intra-peritoneal injection with glucose
(1 g/kg body weight), and then 15, 30, 60, 90, and 120 min after
injection. Plasma glucose concentrations were calculated using a
One Touch ultra-monitoring system (Johnson & Johnson, Co.,
New Brunswick, NJ, USA). The GTT was performed on mice
10 weeks old and repeated when the mice reached 20 weeks of
age and the area under the curve (AUC) for glucose and insulin
were calculated.

PLASMA MEASUREMENTS

Serum was separated from blood samples collected during the
GTT by centrifugation at 3000 rpm for 15 min at 4°C and then
stored in aliquots at — 80°C until assayed. Serum insulin, leptin,
and adiponectin levels were measured in duplicate using commer-
cially available ELISA kits following the manufacturer's protocol
(insulin cat* 90060 and leptin cat* 90030, Crystal Chem, Inc.,
Downers Grove, IL, USA; adiponectin cat* EZMADP-60, Linco
Research, Billerica, MA, USA). Absorbance was read at 450 nm on
a Packard SpectraCount microplate reader (Packard Instrument,
Co.,Meriden, CT,USA).

DIET STUDY

Male heterozygous (RenTgMK +/ ~ ) mice and their WT littermates
were randomly assigned to either a high-fat diet (60% kcal from
fat cat* D12492, Research Diets, Inc., New Brunswick, NJ, USA)
or a low-fat diet (10% kcal from fat cat* D12450B, Research
Diets, Inc., New Brunswick, NJ, USA) for 18 weeks. Each diet
group (n = 6/group) was comprised of three male RenTgMK +/ ~
mice and three male WT mice. Body weight measurements were
acquired weekly for the duration of the study. At the conclusion
of the 18-week diet study, a GTT was performed and plasma
insulin, leptin, and adiponectin concentrations were measured,
as described above. Mice were sacrificed 1 week after the GTT.

METABOLIC STUDIES

Metabolic studies of the RenTgMK mice were performed at the
Mouse Metabolic Phenotyping Center (MMPC) at Vanderbilt
University, Nashville, TN, USA. Glucose infiltration rate, glucose
turnover rate, endogenous glucose turnover rate, and clearance
were measured. Whole-body insulin activity in vivo was exam-
ined via euglycemic hyperinsulinemic clamp. Detailed procedure
has been previously reported (Ayala et al., 2006). Briefly, to
assess insulin sensitivity and glucose metabolism, insulin was con-
tinuously administered via euglycemic hyperinsulinemic clamp.
Catheters were chronically implanted in the jugular vein and
carotid artery. Arterial glucose levels were measured every 5-
10 min during 120 min and glucose infusion rates were deter-
mined based on the arterial glucose measurements. Plasma glucose
turnover was measured in RenTgMK +/ ~ and WT males (« = 8-
12/group). Mice were continuously infused with [3- 3 H]glucose at
a rate of 0.4 ixCi/min. Glucose appearance (Ra) and disappear-
ance (Rd) rates were estimated as the ratio of the rate of infusion
of [3- 3 H]glucose and the steady-state plasma [ 3 H]glucose specific
activity (dpm/mg), and the glucose disappearance was assumed

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Metabolic phenotyping of renin transgenic mice

to be equal to the steady-state Ra rate. Glucose clearance was
calculated by dividing the Rd by the arterial glucose concentra-
tion. To measure tissue-specific glucose uptake, mice were injected
with 12 |xCi of [ 3 H] -labeled 2-deoxyglucose ([2- 3 H] DG). Arterial
plasma samples were collected in intervals for 40 min before mice
were anesthetized and tissues were extracted and frozen in liquid
nitrogen until further analysis.

PANCREAS HISTOLOGY AND IMMUNOSTAINING

The pancreas was collected from WT and transgenic mice. Tissues
for immunohistochemistry were fixed in 10% neutral, phosphate-
buffered formalin for 24 h and paraffin-embedded. Subsequently,
the paraffin-embedded tissues were processed in 4-u,m sections.
Sections were stained using rabbit anti-glucagon polyclonal anti-
body and guinea pig anti-insulin serum (both from Millipore,
Billerica, MA, USA). For fluorescence detection, goat anti-guinea
pig IgG coupled to Texas Red and donkey anti-rabbit IgG cou-
pled to Cy3 were used (both from Jackson ImmunoResearch, West
Grove, PA, USA) followed by Vectashield Mounting Medium with

4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame,
CA, USA) for nuclear staining.

STATISTICAL ANALYSIS

Data were analyzed in SAS (SAS Institute, Inc., Cary, NC, USA)
using a mixed model analysis of variance (http://dawg.utk.edu).
Fisher's test followed by Tukey's post hoc test was used for mean
separation. P < 0.05 was considered statistically significant. Data
are reported as the means ± SE.

RESULTS

BODY WEIGHT. FAT PAD WEIGHT, AND METABOLIC PARAMETERS

Body weights were comparable between mice with either one
or two copies of the renin transgene and WT control mice
(Figure 1A). Gonadal fat pad weight (Figure IB) and adi-
posity index (gonadal fat pad weight divided by body weight;
Figure 1C) were significantly lower in homozygous mice com-
pared to WT littermates (P < 0.05). Fasting serum glucose, leptin,
and adiponectin levels were comparable between all genotypes

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FIGURE 1 | Body and fat pad weight and glucose tolerance in male
RenTgMK mice. (A) Body weight at the age of 20 weeks. (B) Mice
were sacrificed at the end of week 20 and gonadal fat pads were
collected and weighed. (C)The adiposity index was determined by
dividing gonadal fat pad weight by final body weight. A glucose
tolerance test (GTT) was administered after overnight fasting. (D) Blood

glucose levels were measured at 0, 15, 30, 90, and 120 min and plotted
on a graph. (E) Area Under the Curve (AUC) was calculated as
described in the experimental procedures. Values are means ± SE.
n = 6 For WT; n = 5for RenTgMK*'-; n = 4for RenTgM K +/+ . Different
letters indicate a significant difference (P < 0.05). "Significantly
different (P < 0.05) from WT

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Metabolic phenotyping of renin transgenic mice

Table 1 | Serum metabolic markers in male wild-type and RenTgMK
mice.

WT

RenTgMK +/ - RenTgMK +/+ P value

Glucose, mg/dl 81.2±6.4 89.8±3.2 93.3±5.1 0.292
Insulin, ng/ml 0.62±0.07 a 0.42±0.05 b 0.36±0.07 b 0.033
Leptin, ng/ml 2.1 ±0.7 2.3±0.6 1.3±0.5 0.535

Adiponectin, 8.7 ±1.4 10.8 ±2.5 7.6 ±0.9 0.479

jig/ml

C-peptide, 1.4±0.1 b 1.9±0.2 a 2.0±0.1 a 0.007

ng/ml

FFA, mM 0.84±0.10 0.93±0.06 0.91 ±0.11 0.791

Triglycerides, 60.1 ±6.4 a 44.5±8.6 a < b 23.7±8.7 b 0.018
mg/dL

Values are means ± SE. Animals were 21 weeks old. Initial body weight mea-
surements were taken at 10 weeks. Blood was collected after fasting overnight
and metabolic parameters were measured from serum. n=6 For WT; n = 5 for
RenTgMK*'' ; n=4 for RenTgMK*'* . C-peptide, connecting peptide; FFA, free
fatty acid.

Means in a row with superscripts without a common letter differ, P< 0.05.
Numbers in bold indicate a significance of P < 0.05.

(Table 1). Fasting serum insulin, however, was significantly lower
in the transgenic mice (both homozygous and heterozygous) com-
pared to WT littermates (P < 0.05). Serum C-peptide levels, on
the other hand, were higher in the transgenics than in the WT
mice (Table 1 ) . Interestingly, serum triglycerides were significantly
lower in the homozygous mice compared to WT littermates. On
this low-fat chow diet condition, the overall metabolic phenotype
was less pronounced in female transgenic mice compared to WT
female littermates (data not shown).

GLUCOSE INTOLERANCE IN RenTg MICE

To assess glucose tolerance in the RenTg mice, an intra-peritoneal
GTT was administered. Baseline fasting glucose levels were com-
parable between WT, RenTgMK +/ ~ and RenTgMK +/+ mice
(81.17 ± 15.68, 89.80 ±7.16, and 93.25 ± 10.28 mg/dl, respec-
tively). Heterozygous mice maintained significantly higher levels
of glycemia compared to WT within 60 min and remained ele-
vated throughout the GTT (Figure ID). These differences were
observed as early as 10 weeks of age (data not shown) and became
more pronounced with age by 20 weeks. Glucose intolerance in
male RenTgMK mice was also evident from a comparison of the
glucose AUC (Figure IE). The AUC values for both heterozygous
and homozygous mice were higher (P < 0.05) than that of WT
mice implying greater glucose intolerance in the transgenics. In
females, no significant differences in GTT were observed between
the three genotypes at 20 weeks of age in these low-fat feeding
conditions (data not shown).

METABOLIC PHENOTYPING OF RenTg MICE

Insulin resistance is commonly associated with high adiposity.
The paradoxical glucose intolerance despite low adiposity and low
insulinemia in the renin transgenic male mice vs. control litter-
mates led us to further investigate whether these differences were
due to altered insulin sensitivity and/or glucose production or

utilization in this model. Accordingly, metabolic studies at the NIH
MMPC at Vanderbilt University were conducted. Male heterozy-
gous mice were compared to WT mice because males exhibited
glucose intolerance and sufficient numbers could be obtained
from a few litters. Steady-state glucose infusion rate (Figure 2),
overall tissue-specific glucose uptake, glucose metabolism, and
endogenous glucose production (Table 2) did not significantly
differ between RenTgMK and WT mice, indicating normal insulin
sensitivity in the transgenics.

EFFECT OF HIGH-FAT DIET ON BODY WEIGHT, ADIPOSITY, CIRCULATING
ADIP0KINES, AND GLUCOSE TOLERANCE

As described above, renin transgene overexpression led to impaired
glucose tolerance compared to WT mice when mice were fed a
low-fat chow diet. To test whether the genetic differences would be
exacerbated by high-fat feeding, we fed male heterozygous and WT
mice a low- or high-fat diet to investigate diet-gene interactions.

Body weights were not significantly different between groups at
the start of the randomized diet study (Table 3). High-fat feeding
increased body weight only in the wild-type mice (Figure 3A).
Mice of both genotypes showed a trend for increased fat pad
weight and adiposity with high-fat feeding, although the differ-
ence was only significant for adiposity in the RenTgMK +/ ~ mice
(Figures 3B,C).

Changes in adiposity are known to alter hormonal and metabo-
lite levels. As expected, high-fat feeding increased serum glucose
and leptin levels in both male WT and transgenic mice (P < 0.05
for diet effect - Table 3). Interestingly, high-fat feeding also
increased serum resistin levels in WT, but not in transgenic males
(Table 3). In the WT males, serum triglyceride concentration was
higher in the low-fat fed mice when compared to high-fat fed ones
(Table 3). This effect was minimal in the transgenics.

Low-fat fed male heterozygous mice exhibited a higher glucose
excursion and area under the glucose curve compared to their
WT counterparts, indicating glucose intolerance (Figures 3D,E).
High-fat feeding did not exacerbate glucose intolerance in RenT-
gMK mice.

PANCREAS HISTOLOGY AND IMMUNOSTAINING OF RenTg MICE

Because of the consistently lower insulin levels in heterozygous
mice compared to WT mice, we performed immunohistological
studies in the pancreas to assess islet morphology and hormone
content. In both genotypes, islets appeared normal and exhibited
comparable staining for glucagon and insulin (Figure 4).

DISCUSSION

Many lines of evidence have linked activation of the RAS to the
development of obesity and insulin resistance (Schorr et al., 1998;
Van Harmelen et al., 2000), but the effects of increased circu-
lating levels of angiotensins on adiposity remain controversial.
We hypothesized that chronic systemic RAS activation via trans-
genic renin overexpression in the liver would lead to glucose
intolerance and systemic insulin resistance. We also predicted
that increased systemic Ang II would increase adiposity, based
on previous work by us and others showing that Ang II increases
adipocyte lipogenesis and triglyceride storage. Our results demon-
strate that elevated circulating Ang II due to renin overexpression

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Fletcher et al.

Metabolic phenotyping of renin transgenic mice

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FIGURE 2 | Assessment of insulin sensitivity in male wild-type and RenTgMK ' mice using hyperinsulinemic euglycemic clamp. Changes in blood
glucose concentration (A) and glucose infusion rate (B) over time are shown. Values are means± SE. Animals were approximately 9 months old. n = 8-9 For
WT; n = 7forRenTgMK + '-.

Table 2 | Metabolic characteristics and accumulation of [2- 3 H]DG
during the hyperinsulinemic-euglycemic clamp experiments in male
wild-type and RenTgMK +/ ~ mice.

WT

RenTgMK +/ "

P value

Blood glucose, mg/dl

114.7±5.8

114.6±5.0

0.996

GTR, mg/kg/min

47.4 ±4.0

49.4 ±6.7

0.794

endoGTR, mg/kg/min

4.96 ±4.62

-2.29 ±7.60

0.416

Glucose clearance, mg/kg/min

42.0±3.1

43.7 ±5.7

0.790

GIR, mg/kg/min

42.4±4.3

51.7±4.0

0.142

ACCUMULATION OF [2- 3 H]DG

Soleus, u.g/min/mg tissue

0.035 ±0.008

0.036 ±0.005

0.923

Gastro, ^g/min/mg tissue

0.025±0.005

0.033 ±0.003

0.229

Vastus 1., (i,g/min/mg tissue

0.041 ±0.007

0.047 ±0.005

0.550

WAT, (xg/min/mg tissue

0.004 ±0.001

0.006±0.001

0.067

Diaphragm, (ig/min/mg tissue

0.131 ±0.019

0.091 ±0.007

0.103

Heart, |ig/min/mg tissue

0.431 ±0.057

0.320 ±0.043

0.161

Brain, [ig/min/mg tissue

0.048 ±0.005

0.049 ±0.003

0.888

Values are means± SE. Animals were approximately 9 months old. n = 8-9 For
WT; n= 7 for RenTgMK*'' . GTR, glucose turnover rate; endoGTR, endogenous
glucose turnover rate; GIR, glucose infusion rate; [2 J H1DG, 2-deoxy-F Hlglucose;
Gastro, gastrocnemius; Vastus I., vastus lateralis; WAT white adipose tissue.

leads to glucose intolerance, but with consistently lower levels of
plasma insulin. Further, chronic elevation of systemic Ang II by
hepatic overexpression of the renin gene led to a reduction rather

than an increase in adiposity in male mice. However, these mice
exhibit otherwise normal glucose metabolism and a transgene
dose-dependent decrease in insulinemia.

GLUCOSE INTOLERANCE IN RenTgMK MICE

Consistent with previous studies of Ang II infusion and transgenic
renin expression (Lee et al., 1996), male RenTgMK transgenic mice
exhibited glucose intolerance, even on a low-fat diet. However,
despite this glucose intolerance, the RenTgMK mice maintained
low fasting insulinemia and normal insulin sensitivity, as indicated
by normal steady-state glucose infusion during the hyperinsuline-
mic, euglycemic clamp studies. RenTgMK mice maintained low
insulinemia even under high-fat feeding. The glucose intolerance
in RenTgMK mice in the presence of normal fasting glucose levels
and low insulinemia, a feature that is a rather typical hallmark
of increased insulin sensitivity, could be due to decreased insulin
production/secretion and/or increased insulin clearance. Serum
C-peptide level was higher in heterozygous compared to WT mice
arguing against decreased insulin secretion accounting for low
insulinemia in the RenTgMK mice. Immunohistochemistry of
the pancreas indicated normal islet morphology and hormone
content, possibly indicating normal pancreatic function. How-
ever, such studies are only qualitative and do not allow to detect
clear quantitative differences. Thus, it is probably insulin clearance,
rather than insulin secretion that may be altered in this model.

Liver is the primary site of insulin clearance (Duckworth
et al., 1998), which can be affected by both nutritional and hor-
monal signals. Insulin clearance rate is heritable (Goodarzi et al,

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Metabolic phenotyping of renin transgenic mice

Table 3 | Effects of high-fat diet on body weight and metabolic characteristics in male wild-type and RenTgMK ' mice.

WT RenTgMK +/ - P value

LF HF LF HF Geno Diet Geno X diet

Initial body weight, g

27.2 ±2.2

29. 2± 1.4

26.2 ±0.6

25.7±0.9

NS

NS

NS

Final body weight, g

37.6±3.2 a ' b

46.9±5.6 a

35.4± 1.8 b

37.8 ± 2.1 a ' b

NS

NS

NS

Glucose, mg/dL

92.3±3.2 b

123.7±7.7 a

98.3±5.2 b

127.3±2.9 a

NS

0.001

NS

Insulin, ng/ml

1.29±0.31

2.07±0.60

0.91 ±0.04

1.14±0.25

NS

NS

NS

Leptin, pg/ml

9.5±1.6

16.2±2.9

7.6 ±1.1

12.7±2.3

NS

0.019

NS

Adiponectin, ng/ml

12.6±0.3

14.1 ±2.0

12.9±0.4

12.4±0.2

NS

NS

NS

Resistin, pg/ml

475.8 ±37.3 b

702.2 ±19.0 a

545.8 ± 48. 7 a ' b

493.3 ±83.9 b

NS

NS

0.030

MCP-1, pg/ml

29.9±8.0

53. 0± 18.9

30.5±1.6

34.0±12.9

NS

NS

NS

PAI-1 , pg/ml

3903.4 ±666.2

5903.4 ±834.8

4733.5 ±734.0

4526.8±961.3

NS

NS

NS

C-peptide, ng/ml

1.9±0.3

3.3±1.2

2.4±0.4

2.3±0.2

NS

NS

NS

FFA, mM

1.00±0.18

0.80 ±0.04

1.23 ±0.36

1.16±0.39

NS

NS

NS

Triglycerides, mg/dL

132.4±16.5 a

66.9 ± 4. 1 b

66.8±4.4 b

39.2±5.4 b

0.001

0.001

0.074

Values are means ± SE. Animals were fed a high-fat or low-fat diet for 19 weeks. Initial body weight measurements were taken at the beginning of the study. Mice
were 3-5 months old. Blood was collected after fasting overnight and metabolic parameters were measured from serum. n = 3 For each group. LF low-fat; HF high-fat;
MCP-1, monocyte chemoattractant protein-1; PAI-1, plasminogen activator inhibitor-1; C-peptide, connecting peptide; FFA, free fatty acid.
Means in a row with superscripts without a common letter differ, P< 0.05.
Numbers in bold indicate a significance of P < 0.05.

2005) and is reduced in obesity and Type-2 diabetes (Duck-
worth et al., 1998). Therefore, it could be an important factor
in the pathogenesis of Type-2 diabetes. Conversely, there are
mouse models which exhibit increased insulin clearance such as
the mouse overexpressing carcinoembryonic antigen-related cell
adhesion molecule 1 (CEACAM 1 ) in the liver (Najjar, 2002) . Addi-
tional studies beyond the scope of this work will be required
to address whether the RAS is involved in regulating insulin
clearance.

The finding that the glucose intolerance in male transgenic mice
did not worsen with high-fat feeding could possibly indicate that
RAS overactivation could at least in part play a role in high-fat diet-
induced obesity. Along the same lines, mice overexpressing ACT
in adipose tissue also develop glucose intolerance on a low- fat diet,
which is not further exacerbated by high-fat feeding (Kalupahana
et al., 2012). Female transgenic mice exhibited normal glucose tol-
erance on a low-fat diet while males became glucose intolerant on
the same diet when compared to WT littermates. Further, female
transgenics became glucose intolerant when fed a high-fat diet
(data not shown).

It is likely that the metabolic phenotype of the RenTgMK mice
is due to Ang II effects, rather than the effects of renin acting
on the renin/prorenin receptor. We argue this because in renin
knockout mice, the metabolic phenotype of increased insulin sen-
sitivity and resistance to high-fat diet-induced glucose intolerance
and insulin resistance was reversed by Ang II infusion (Takahashi
et al., 2007). It is also likely that these effects are mediated via
angiotensin receptors, as previous studies on the RenTgMK mice
demonstrated that ATI receptor blockade reversed renal pathol-
ogy and normalized blood pressure in the RenTgMK mice (Caron
et al, 2002). Alternative mechanisms may involve direct effects of
renin mediated by the renin/prorenin receptor on the vasculature
or adipose tissue. Indeed, renin receptors are expressed in adipose

tissue (Achard et al, 2007) and therefore may mediate the observed
adipose tissue phenotype.

RAS OVERACTIVATION AND INSULIN RESISTANCE

Renin-angiotensin system overactivation via chronic Ang II infu-
sion leads to the development of systemic insulin resistance
in rodents. This is, in most part, due to the Ang II-mediated
impairment of skeletal muscle glucose transport and utilization
(Kalupahana and Moustaid-Moussa, 2012a). Ang II impedes the
insulin-mediated tyrosine phosphorylation of the insulin receptor
substrate (IRS)-l, activation of Akt, and translocation of glucose
transporter (Glut) -4 in the skeletal muscle in an NADPH oxi-
dase, ATI, and NF-kB-dependent manner. Ang II also increases
hepatic glucose production, which also potentially contributes to
altered systemic insulin sensitivity. In contrast, the RenTgMK mice
in this study exhibited normal systemic insulin sensitivity. While
the exact underlying mechanisms for this discrepancy of insulin
sensitivity between different models of RAS overactivation are
unknown, it is possible that the low insulinemia present in the
RenTgMK mice could protect these mice from the development
of insulin resistance. Previous studies have shown that an increase
in plasma insulin by itself can induce insulin resistance. In the
study by Shanik et al. (2008), mice transfected with extra copies
of the insulin gene had a two- to four-fold increase in plasma
insulin and exhibited normal body weight, insulin resistance and
hypertriglyceridemia.

Unlike models of chronic Ang II infusion (Ran et al, 2004),
RenTgMK mice exhibited lower plasma triglyceride levels. Thus,
the hypoinsulinemia in the RenTgMK could also potentially
explain the low serum triglyceride levels seen in these mice. Given
this metabolic phenotype of RenTgMK mice, it would be interest-
ing to explore whether the insulin resistance seen in several models
of chronic RAS overactivation is insulin-dependent and further

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Fletcher et al.

Metabolic phenotyping of renin transgenic mice

25 n
20
5 5 -

J3

60

4 •

5
0

□ LF
■ HF

•g q

WT

RenTgMK+/-

WT

RenTgMK+/-

WT

RenTgMK+/-

600
500
400
300
200
100
0

-^WT (LF)
-A-WT (HF)
-O- RenTgMK+/ - (LF)
-■- RenTgMK+/- (HF)

1 1 r

15 30 60

Time (minutes)

120

70000 i

60000

3 50000
<

§ 40000
° 30000

20000

10000

WT

RenTgMK+/-

FIGURE 3 | Effect of high-fat diet on body and fat pad weight and
glucose tolerance of male wild-type and RenTgMK +/ " mice. (A) Weight
gain was calculated as the difference between the initial body weight
measured at week 1 and the final weight measured after 1 8 weeks. (B) Mice
were sacrificed at the end of week 19 and gonadal fat pads were collected
and weighed. (C)The adiposity index was determined by dividing gonadal fat

pad weight by final body weight X 100. (D) A glucose tolerance test (GTT) was
administered after overnight fasting. Blood glucose levels were measured at
0, 15, 30, 90, and 120 min and plotted on a graph. (E) Area Under the Curve
(AUC) was calculated as described in the experimental procedures. Values are
means ± SE. n = 3 For each group. "Significantly different (P < 0.05) from
WT-LF Different letters indicate a significant difference (P < 0.05).

studies are warranted. The issue of whether the renin recep-
tor may also in part modulate insulin sensitivity merits further
investigation as well.

RAS 0VERACTIVATI0N AND ADIPOSITY

Both human and rodent studies have shown that obesity and
increased adiposity are associated with both systemic and adipose
RAS overactivation (Kalupahana and Moustaid-Moussa, 2012a).
However, it is not known whether primary RAS overactivation
leads to obesity. Transgenic mouse models clearly demonstrate that
manipulating components of the RAS alters adiposity: mice over-
expressing AGT in adipose tissue have increased adiposity, while
deletion of either the AGT or Ang II receptor genes reduces fat-
ness. Paradoxically, previous studies of chronic Ang II infusion in
rodents have shown that chronic systemic RAS overactivation leads
to weight loss, rather than weight gain (Griffin et al., 1991; Cassis
et al, 1998). The transgenic TGR(mREN2)27 rat overexpressing
the mouse Ren2 renin gene also has a lean phenotype (Mullins
et al, 1990; Langheinrich et al, 1996; Lee et al, 1996). Similar to

these findings, the RenTgMK mice also exhibited lower fat mass
compared to WT littermates. The adipose mass was significantly
decreased by the renin transgene in a gene dosage-dependent
manner. In contrast, mice with primary AGT overproduction in
adipose tissue exhibit higher adiposity (Massiera et al., 2001a).
Further, deletion of AGT and other RAS genes leads to lower
fat mass and resistance to diet-induced obesity (Massiera et al,
2001b; Takahashi et al., 2007). Thus, it appears that while sys-
temic RAS overactivation leads to reductions in body weight,
local increases in RAS activity in adipose tissue leads to increased
adiposity.

The low-fat mass observed following Ang II infusion is attrib-
uted to both increased energy expenditure and reduced energy
intake (Brink et al, 1996; Cassis et al., 1998). In the RenTgMK
mouse model, we did not detect any significant differences in food
intake (data not shown). Activation of the sympathetic nervous
system may also account for changes in weight via modulation
of lipid metabolism and energy expenditure by catecholamines
(Cassis, 2000). The differential effect of systemic vs. adipose

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January 2013 | Volume 3 | Article 166 | 7

Fletcher et a

Metabolic phenotyping of renin transgenic mice

FIGURE 4 | Islet pathology. Pancreas histology and immunostaining were conducted to assess islet morphology and hormone content in male wild-type and
RenTgMK mice, 20 weeks of age.

specific RAS overactivation on adiposity indicates that specific
local overproduction of AGT in adipose tissue per se, may be
required for increasing adiposity. Indeed, Ang II exerts local ana-
bolic effects in the adipose tissue (Massiera et al, 2001a). Ang
II also increases lipogenic gene expression and enzyme activ-
ity in 3T3-L1 murine adipocytes and human adipocytes in vitro
(Jones et al., 1997). This is also in agreement with studies showing
differentiation-dependent increase in AGT gene expression and
secretion in preadipocytes (Kim and Moustaid-Moussa, 2000).
Ubiquitous inactivation of AGT, on the other hand, results in
significant loss of fat mass. However, it is unclear whether tar-
geted inactivation of AGT in adipose tissue would specifically
alter fat mass and such studies would convincingly confirm
the role of adipose AGT in modulating insulin resistance or
fat mass.

In summary, our data demonstrate that transgenic hepatic over-
expression of renin leads to glucose intolerance, decreased fat mass,

hypoinsulinemia, and hypotriglyceridemia, with normal systemic
insulin sensitivity. The hypoinsulinemia in these mice is possi-
bly due to increased insulin clearance, as indicated by elevated
C-peptide levels and normal pancreatic insulin levels indicating
normal pancreatic function. Whether the unexpected low adipos-
ity and normal insulin sensitivity despite the presence of glucose
intolerance in the RenTgMK mice is secondary to hypoinsulinemia
merits further investigation.

ACKNOWLEDGMENTS

The authors thank Dr. Oliver Smithies for generously providing us
with the RenTg mice we used to perform this study and Dr. K. Car-
ron and J. Hagaman for providing help with animal breeding and
genotyping. This work was supported by a USDA NIFA-NRI Grant
2005-35200-15224 and an AHA Grant in Aid (Greater Southeast
affiliate 0755626B). The authors would like to thank Jeffrey Morris
and Taryn Stewart for their technical assistance.

REFERENCES

Achard, V., Boullu-Ciocca, S., Desbriere,
R., Nguyen, G., and Grino, M.
(2007). Renin receptor expression in
human adipose tissue. Am. J. Phys-
iol. Regul. Integr. Comp. Physiol. 292,
R274-R282.

Ayala, J. E., Bracy, D. R, McGuin-
ness, O. R, and Wasserman, D.
H. (2006). Considerations in the
design of hyperinsulinemic-
euglycemic clamps in the
conscious mouse. Diabetes 55,
390-397.

Bosch, J., Yusuf, S., Gerstein, H. C,
Pogue, J., Sheridan, R, Dagenais, G.,
et al. (2006). Effect of ramipril on
the incidence of diabetes. N. Engl. J.
Med 355, 1551-1562.

Brink, M., Wellen, J., and Delafontaine,
R (1996). Angiotensin II causes
weight loss and decreases circulat-
ing insulin-like growth factor I in
rats through a pressor-independent
mechanism. /. Clin. Invest 97,
2509-2516.

Caron, K. M., James, L. R., Kim, H.
S., Knowles, J„ Uhlir, R., Mao, L.,

et al. (2004). Cardiac hypertrophy
and sudden death in mice with a
genetically clamped renin transgene.
Proc. Natl. Acad. Sci. U.S.A. 101,
3106-3111.

Caron, K. M., James, L. R., Kim, H. S.,
Morham, S. G., Sequeira Lopez, M.
L., Gomez, R. A., et al. (2002). A
genetically clamped renin transgene
for the induction of hypertension.
Proc. Natl. Acad. Sci. U.S.A. 99,
8248-8252.

Cassis, L. A. (2000). Fat cell metab-
olism: insulin, fatty acids, and

renin. Curr. Hypertens. Rep. 2,
132-138.

Cassis, L. A., Marshall, D. E., Fettinger,
M. J., Rosenbluth, B., and Lodder, R.
A. (1998). Mechanisms contributing
to angiotensin II regulation of body
weight. Am. J. Physiol. 274(Pt 1),
E867-E876.

Duckworth, W. C, Bennett, R. G.,
and Hamel, F. G. (1998). Insulin
degradation: progress and potential.
Endocr. Rev. 19, 608-624.

Goodarzi, M. O., Taylor, K. D., Guo,
X., Quinones, M. J., Cui, J., Li, X.,

Frontiers in Endocrinology | Diabetes

January 2013 | Volume 3 | Article 166 | 8

Fletcher et al.

Metabolic phenotyping of renin transgenic mice

et al. (2005). Variation in the gene
for muscle-specific AMP deaminase
is associated with insulin clearance,
a highly heritable trait. Diabetes 54,
1222-1227.

Griffin, S. A., Brown, W. C, MacPher-
son, E, McGrath, J. C, Wil-
son, V. G., Korsgaard, N., et al.
(1991). Angiotensin II causes vas-
cular hypertrophy in part by a
non-pressor mechanism. Hyperten-
sion 17, 626-635.

Hatada, S., Kuziel, W., Smithies, O.,
and Maeda, N. (1999). The influ-
ence of chromosomal location on
the expression of two transgenes in
mice. /. Biol. Chem. 274, 948-955.

Jayasooriya, A. P., Mathai, M. L.,
Walker, L. L., Begg, D. P., Denton,
D. A., Cameron-Smith, D., et al.
(2008). Mice lacking angiotensin-
converting enzyme have increased
energy expenditure, with reduced fat
mass and improved glucose clear-
ance. Proc. Natl. Acad. Sci. U.S.A.
105, 6531-6536.

Jeunemaitre, X. (2008). Genetics of the
human renin angiotensin system. /
Mol. Med. (Bed.) 86, 637-641.

Jeunemaitre, X., Gimenez-Roqueplo,
A. P., Celerier, J., and Corvol,
P. (1999). Angiotensinogen vari-
ants and human hypertension. Curr.
Hypertens. Rep. 1, 31-41.

Jones, B. H., Standridge, M. K., and
Moustaid, N. (1997). Angiotensin II
increases lipogenesis in 3T3-L1 and
human adipose cells. Endocrinology
138, 1512-1519.

Kalupahana, N. S., Massiera, E,
Quignard-Boulange, A., Ailhaud,
G, Voy, B. H., Wasserman, D. H.,
et al. (2012). Overproduction of
angiotensinogen from adipose tis-
sue induces adipose inflammation,
glucose intolerance, and insulin
resistance. Obesity (Silver Spring)
20,48-56.

Kalupahana, N. S., and Moustaid-
Moussa, N. (2012a). The renin-
angiotensin system: a link between
obesity, inflammation and insulin
resistance. Obes. Rev. 13, 136-149.

Kalupahana, Nishan S., and Moustaid-
Moussa, N. (2012b). The adipose

tissue renin-angiotensin system and
metabolic disorders: a review of
molecular mechanisms. Crit. Rev.
Biochem. Mol. Biol. 47, 379-390.
Kim, S., and Moustaid-Moussa, N.
(2000). Secretory, endocrine and
autocrine/paracrine function of the
adipocyte. /. Nutr. 130, 3110S-
3115S.

Langheinrich, M., Lee, M. A., Bohm,
M., Pinto, Y. M., Ganten, D., and
Paul, M. (1996). The hyperten-
sive Ren-2 transgenic rat TGR
(mREN2)27 in hypertension
research. Characteristics and func-
tional aspects. Am. J. Hypertens. 9,
506-512.

Lee, M. A., Bohm, M., Paul, M., Bader,
M., Ganten, U., and Ganten, D.
(1996). Physiological characteriza-
tion of the hypertensive transgenic
rat TGR(mREN2)27. Am. J. Physiol.
270(Pt 1),E919-E929.

Lee, M. H., Song, H. K., Ko, G. J.,
Kang, Y. S., Han, S. Y, Han, K. H,
et al. (2008). Angiotensin receptor
blockers improve insulin resistance
in type 2 diabetic rats by modulat-
ing adipose tissue. Kidney Int. 74,
890-900.

Massiera, F., Bloch-Faure, M., Ceiler,
D., Murakami, K., Fukamizu, A.,
Gasc, J. M., et al. (2001a). Adi-
pose angiotensinogen is involved in
adipose tissue growth and blood
pressure regulation. FASEB J. 15,
2727-2729.

Massiera, E, Seydoux, J., Geloen,
A., Quignard-Boulange, A., Tur-
ban, S., Saint-Marc, P., et al.
(2001b). Angiotensinogen-deficient
mice exhibit impairment of diet-
induced weight gain with alter-
ation in adipose tissue development
and increased locomotor activity.
Endocrinology 142, 5220-5225.

Mullins, J. J., Peters, J., and Ganten,
D. (1990). Fulminant hypertension
in transgenic rats harbouring the
mouse Ren-2 gene. Nature 344,
541-544.

Najjar, S. M. (2002). Regulation of
insulin action by CEACAM1.
Trends Endocrinol. Metab. 13,
240-245.

Ogihara, T, Asano, T, Ando, K.,
Chiba, Y, Sakoda, H., Anai, M., et
al. (2002). Angiotensin Il-induced
insulin resistance is associated with
enhanced insulin signaling. Hyper-
tension 40, 872-879.

Ran, J., Hirano, T, and Adachi, M.
(2004). Chronic ANG II infu-
sion increases plasma triglyceride
level by stimulating hepatic triglyc-
eride production in rats. Am. J.
Physiol. Endocrinol. Metab. 287,
E955-E961.

Schmieder, R. E., Hilgers, K. E,
Schlaich, M. P., and Schmidt, B.
M. (2007). Renin-angiotensin sys-
tem and cardiovascular risk. Lancet
369, 1208-1219.

Schorr, U., Blaschke, K., Turan, S-,
Distler, A., and Sharma, A. M.
(1998). Relationship between
angiotensinogen, leptin and blood
pressure levels in young nor-
motensive men. /. Hypertens. 16,
1475-1480.

Shanik, M. H.,Xu,Y., Skrha, J., Dankner,
R., Zick, Y, and Roth, J. (2008).
Insulin resistance and hyperinsu-
linemia: is hyperinsulinemia the
cart or the horse? Diabetes Care
31(Suppl. 2), S262-S268.

Takahashi, N., Li, E, Hua, K., Deng, J.,
Wang, C. H, Bowers, R. R., et al.
(2007). Increased energy expendi-
ture, dietary fat wasting, and resis-
tance to diet-induced obesity in
mice lacking renin. Cell Metab. 6,
506-512.

Truett, G. E., Heeger, P., Mynatt, R. L.,
Truett, A. A., Walker, J. A., and War-
man, M. L. (2000). Preparation of
PCR-quality mouse genomic DNA
with hot sodium hydroxide and tris
(HotSHOT). Biotechniques 29, 52,
54.

Van Harmelen, V, Ariapart, P., Hoffst-
edt, J., Lundkvist, I., Bringman, S.,
and Arner, P. (2000). Increased adi-
pose angiotensinogen gene expres-
sion in human obesity. Obes. Res. 8,
337-341.

Vermes, E., Ducharme, A., Bourassa,
M. G, Lessard, M., White, M., and
Tardif, J. C. (2003). Enalapril reduces
the incidence of diabetes in patients

with chronic heart failure: insight
from the Studies Of Left Ventricular
Dysfunction (SOLVD). Circulation
107, 1291-1296.

Xiao, E, Wei, H., Song, S., Li, G, and
Song, C. (2006). Polymorphisms
in the promoter region of the
angiotensinogen gene are associated
with liver cirrhosis in patients with
chronic hepatitis B. /. Gastroenterol.
Hepatol. 21, 1488-1491.

Yvan-Charvet, L., Even, P., Bloch-Faure,
M., Guerre-Millo, M., Moustaid-
Moussa, N., Ferre, P., et al. (2005).
Deletion of the angiotensin type 2
receptor (AT2R) reduces adipose cell
size and protects from diet-induced
obesity and insulin resistance. Dia-
betes 54, 991-999.

Conflict of Interest Statement: The

authors declare that the research was
conducted in the absence of any com-
mercial or financial relationships that
could be construed as a potential con-
flict of interest.

Received: 04 November 2012; accepted:
02 December 2012; published online: 07
January 2013.

Citation: Fletcher SJ, Kalupahana NS,
Soltani-Bejnood M, Kim JH, Saxton
AM, Wasserman DH, De Taeye B,
Voy BH, Quignard-Boulange A and
Moustaid-Moussa N (2013) Transgenic
mice overexpressing renin exhibit glu-
cose intolerance and diet-genotype inter-
actions. Front. Endocrin. 3:166. doi:
10.3389/fendo.2012.00166
This article was submitted to Frontiers
in Diabetes, a specialty of Frontiers in
Endocrinology.

Copyright © 2013 Fletcher, Kalupahana,
Soltani-Bejnood, Kim, Saxton, Wasser-
man, De Taeye, Voy, Quignard-Boulange
and Moustaid-Moussa. This is an open-
access article distributed under the terms
of the Creative Commons Attribution
License, which permits use, distribution
and reproduction in other forums, pro-
vided the original authors and source
are credited and subject to any copy-
right notices concerning any third-party
graphics etc.

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